The role of ion homeostasis in adaptation and tolerance to acetic acid stress in yeasts

Abstract Maintenance of asymmetric ion concentrations across cellular membranes is crucial for proper yeast cellular function. Disruptions of these ionic gradients can significantly impact membrane electrochemical potential and the balance of other ions, particularly under stressful conditions such as exposure to acetic acid. This weak acid, ubiquitous to both yeast metabolism and industrial processes, is a major inhibitor of yeast cell growth in industrial settings and a key determinant of host colonization by pathogenic yeast. Acetic acid toxicity depends on medium composition, especially on the pH (H+ concentration), but also on other ions’ concentrations. Regulation of ion fluxes is essential for effective yeast response and adaptation to acetic acid stress. However, the intricate interplay among ion balancing systems and stress response mechanisms still presents significant knowledge gaps. This review offers a comprehensive overview of the mechanisms governing ion homeostasis, including H+, K+, Zn2+, Fe2+/3+, and acetate, in the context of acetic acid toxicity, adaptation, and tolerance. While focus is given on Saccharomyces cerevisiae due to its extensive physiological characterization, insights are also provided for biotechnologically and clinically relevant yeast species whenever available.


Introduction
Yeast cells must have the ability to quic kl y and effectiv el y r espond to sudden environmental challenges to ensure their fitness and surviv al, a tr ait extensiv el y explor ed in the yeast model and cell factory Sacc harom yces cerevisiae (Estruc h 2000, López-Maury et al. 2008, Saini et al. 2018 ).Centr al to yeast's str ess r esponse is the maintenance of ionic homeostasis, where dissimilarities in ion concentr ations acr oss cellular membr anes establish crucial gr adients for proper cellular function (Orij et al. 2011, Ke et al. 2013, Yenush 2016, Antunes et al. 2023 ).T hese ionic gradients , which define the tr ansmembr ane electr oc hemical potential, ar e fundamental for maintaining cellular physiology, solute translocation, and energy homeostasis, preventing toxicity from intracellular sur plus ion concentr ations (Canadell and Ariño 2016 ).Perturbations to any of these gr adients, especiall y under str ess, can substantially disrupt the overall electrochemical transmembrane potential and the balance of other ions, underscoring the interconnectedness of ion homeostasis, cellular function, and stress tolerance (Orij et al. 2011 ).Despite significant pr ogr ess in understanding yeast ion fluxes and homeostasis, knowledge ga ps r emain r egarding r egulatory mec hanisms, their intr acellular distribution, and the interplay of different ions within the cell, especially under stress conditions.
Acetic acid constitutes a prime example of an environmental stress factor that directly challenges ion homeostasis (Macpherson et al. 2005, Mira et al. 2010b, Wan et al. 2015, Palma et al. 2018, Xu et al. 2020, Guar a gnella and Bettiga 2021 ).This short-chain weak organic acid, despite being a carbon source for several yeast species, is a toxic byproduct of alcoholic fermentation and a widely used food preservative (Mira et al. 2010b, Palma et al. 2018, Cunha et al. 2019 ).The impact of its effects extends to its pr e v alence as a major inhibitor in various industrial settings, and to the pathogenicity of yeast species (Cunha et al. 2017, 2019, Ullah et al. 2013b, Deparis et al. 2017 ).In industrial settings, acetic acid drives the need for the pursuit and engineering of robust str ess-toler ant yeast species to enhance bioproduct and biomass yields, particularly as demand grows for sustainable and green chemicals (Deparis et al. 2017 ).Moreover, understanding yeast responses to acetic acid is crucial in the context of inv asiv e fungal infections, where the ability to withstand this stress can influence pathogenic Candida species ability to thrive and survive during host colonization (Moosa et al. 2004, Ullah et al. 2013b, Cunha et al. 2017, Lourenco et al. 2019 ).
The regulation of intracellular pH (pHi) is a paradigmatic example of the critical nature of ion homeostasis in yeast response to acetic acid-induced to xicity, highly de pendent on the medium pH (i.e.H + concentration) (Orij et al. 2011, Stratford et al. 2013b, Ullah et al. 2013b, Antunes et al. 2023 ).Low pH values in the medium enhance the effectiveness of micr obial gr owth inhibition by acetic acid (Pampulha and Loureiro-Dias 1989, Mira et al. 2010b, Palma et al. 2018 ).This effect results from the balance between the protonated and dissociated forms of acetic acid: as the medium pH decreases from the acetic acid pKa value of 4.75, the concentration of undissociated acetic acid (CH 3 COOH) toxic form incr eases (Arnebor g et al. 2000 ).Notably, acetic acid possesses an inter esting featur e: it can act as both a hydr ogen bond donor and acceptor, enabling the formation of stable dimeric complexes in nonpolar solvents (Pem et al. 2023 ).This c har acteristic pr esumably allows for passive diffusion of the undissociated acid in the form of dimeric complexes across the nonpolar interior of yeast plasma membrane lipids.Acetic acid uptake was also proposed to be facilitated by the aqua gl ycer oporin Fps1 (Mollapour and Piper 2007 ).Raising the medium pH above the pKa value of acetic acid leads to a higher abundance of acetate anions.In Cr abtr eepositi ve yeast species, lik e S. cerevisiae , when acetic acid is the sole carbon source or when glucose or another r epr essing carbon source is absent, these c har ged counterions can enter the cell via a H + -symport mechanism mediated by secondary active monocarboxylic acid tr ansporters suc h as Ady2 and Jen1 (Casal et al. 1996(Casal et al. , 2016 ) ).
Upon encountering a near-neutral pH in the cytosol, acetic acid dissociates into the acetate anion (CH 3 COO − ), accompanied by pr oton r elease (Arnebor g et al. 2000, Mir a et al. 2010b, P alma et al. 2018 ).Since the dissociated acid form cannot escape to the extracellular medium through passive diffusion due to its charge, this leads to its intracellular accumulation, interfering with cellular metabolism and causing o xidati v e str ess (Almeida et al. 2009, Semch ysh yn et al. 2011 ).On the other hand, increased intracellular pr oton concentr ation leads to cytosol acidification and dissipation of the tr ansmembr ane pr oton gr adient (P ampulha and Lour eir o-Dias 1990 , Ding et al. 2013 ).To mitigate the effects of acetic acid stress, yeast cells trigger the ATP-dependent efflux of protons and acetate from the cell (Carmelo et al. 1997, Sá-Correia and Godinho 2022, Zhang et al. 2022 ).Ho w e v er, this activ e efflux results in a significant depletion of the cellular ATP pools, reflected in the reduction in yeast maximum biomass yield and specific growth rate observed under acetic acid stress (Pampulha and Lour eir o-Dias 2000 ).Mor eov er, cellular env elope r earr angements, including alterations in cell wall structure and plasma membr ane lipid composition, ar e crucial to regulate the stability and function of r ele v ant tr ansport pr oteins and, to r educe the rate of diffusion of the undissociated acid form back into the cell.This effectiv el y counter acts the futile cycle created by the active efflux of acetate and protons if follo w ed b y the re-entry of the liposoluble acid form (Van Der Rest et al. 1995, Ullah et al. 2013a, Ribeir o et al. 2021 ).Additionall y, the pr esence and natur e of nutrients (carbon and nitrogen sources, amino acids, and vitamins) and minerals (such as K + , Fe 2 + /3 + , and Zn 2 + ) also contributes to yeast adaptation to acetic acid-induced stress (Macpherson et al. 2005, Mira et al. 2010b, Wan et al. 2015, Zhang et al. 2017, Xu et al. 2019b, Castillo-Plata et al. 2022, Chen et al. 2022, Antunes et al. 2023 ).
This r e vie w explor es the physiological mec hanisms behind ion homeostasis in the context of acetic acid toxicity, adaptation, and toler ance.It pr ovides an ov ervie w of the roles and regulation of the balance and fluxes across cellular membranes of k e y ions involved in the corresponding stress response, such as protons (H + ), potassium (K + ), iron (Fe 2 + /3 + ), zinc (Zn 2 + ), and acetate (CH 3 COO − ).By examining the genes and pr oteins involv ed in str ess-signaling pathways that mediate these ions' r egulation, this r e vie w aims to elucidate the complex strategies emplo y ed b y y easts to maintain ion balance and ensure efficient adaptation and survival under acetic acid stress .T his information is extensive for S. cerevisiae since the molecular mechanisms underlying the response to this stress have been extensively characterized in this species (Palma et al. 2018 , Guaragnella andBettiga 2021 ).Ho w ever, whene v er av ailable, this r e vie w also incor por ates and provides insights on the topic in r ele v ant biotec hnological and clinical yeast species.Two accompan ying figur es ar e pr o vided, displa ying an o v ervie w of acetic acid toxicity mechanisms (Fig. 1 ) and adaptation strategies (Fig. 2 ) in S. cerevisiae from the context of intracellular ion balances and fluxes.

Homeostasis and regulation of proton (H + ) fluxes and the response and tolerance to acetic acid stress
The plasma membrane and organellar H + -ATPases as the main regulators of H + fluxes and homeostasis Plasma membr ane pr oton gr adient in yeast is generated by the activity of plasma membr ane H + -ATP ase Pma1 (Morsomme et al. 2000, Kane 2016, Zhao et al. 2021 ).This H + gradient serves as a primary driving force supporting active transport of ions, nutrients, and metabolites through H + -coupled transporters, which use this gradient to move solutes against their concentration gradients (Kane 2016 ).PMA1 is an essential gene; mutations that result in defects in Pma1 activity commonly lead to reduced specific gr owth r ates and r esistance to hygr omycin B, an indicator of plasma membrane depolarization (Serrano et al. 1986, Perlin et al. 1988 ).The active H + pumping activity of Pma1 accounts for a significant portion of cellular ATP consumption during growth on glucose and under acetic acid stress (Pampulha and Loureiro-Dias 2000 , Ullah et al. 2013a ).Pma1 is the main modulator of cytosolic pH (Serrano et al. 1986, Carmelo et al. 1997 ).Intracellular pH homeostasis is ac hie v ed thr ough the continuous regulation of both cytosolic and organellar pH levels (Canadell and Ariño 2016 ).The pH within any given organelle is determined by a complex interpla y in volving passiv e pr oton leaks alongside the function of various pumps and channels (Brett et al. 2005, Kane 2016, Ariño et al. 2019 ).This regulation of organelle pH is crucial not only for maintaining the internal environment of the organelle but also plays a role in the broader cellular dynamics, including vesicle tr affic king among or ganelles within the secr etory and endosomal pathways (Brett et al. 2005, Kane 2016 ).
Another notable player in pHi homeostasis is the vacuolar-type pr oton-tr anslocating ATP ases (V-ATP ases).They consist of multisubunit enzyme complexes divided into two main sectors: V 1 and V 0 , each composed of several subunits (Kane 2007 ).The V 1 sector, facing the cytoplasm, harbors the catalytic ATPase activity, while the V 0 sector, embedded in the membr ane of or ganelles (v acuole/l ysosomes , endosomes , and Golgi) is responsible for proton translocation (Kane 2007 ).As ATP-driven proton pumps, their primary function involves transporting protons from the cytoplasm into these or ganelles (r e vie wed in Kane 2007 ).This proton transport is crucial for maintaining the acidic pH of diverse organelles, facilitating various cellular processes, including protein sorting, nutrient stor a ge, and detoxification.Remarkabl y, unlike most eukaryotes, deletion of genes coding for any of the subunits or assembly factors ( vma mutants) in S. cerevisiae does not lead to lethality under nonstressing conditions.Ho w ever, it renders cells extr emel y sensitiv e to pH fluctuations, unable to gr ow on nonfermentable carbon sources, and susceptible to stress induced by a wide range of inhibitors, in particular acetic acid and other weak acids, and heavy metals (Kawahata et al. 2006, Kane 2007, Mira et al. 2010b, Tarsio et al. 2011, Deprez et al. 2021, Mota et al. 2024 ).  1 .

Regulation of Pma1 under acetic acid stress
The essential protein Pma1 is composed of 10 tr ansmembr ane helices and three cytosolic domains: the actuator domain (A), the nucleotide binding domain (N), and the phosphorylation domain (P).Furthermore, the C-terminal tail of the H + -ATPase, known as the regulatory (R) domain, plays a crucial role in modulating Pma1 activity (Kane 2016, Zhao et al. 2021 ).This domain can interact with the P domain to autoinhibit the H + -pump activity (Kane 2016, Zhao et al. 2021 ).Pma1 is highly abundant in the plasma membr ane, r epr esenting at least 15% of all plasma membrane proteins (Serrano 1988 ).It has been shown to oligomerize, forming hexamers, a process influenced by sphingolipids and to not be homogeneously distributed across the PM but instead accumulating in a membrane subcompartment denominated MCP (membrane compartment containing Pma1) (Zhao et al. 2021 ).This MCP is c har acterized by being depleted in er goster ol and exclusiv el y enric hed in sphingolipids (Zhao et al. 2021 ).
The activation of Pma1 is among the cellular responses when cells are exposed to acetic acid stress (Carmelo et al. 1997, Stratford et al. 2013a, Antunes et al. 2023 ).Under this stress, Pma1 activ el y pumps pr otons out of the cell, helping to r estor e pHi up to more physiological values and preventing further acidification and potential harm to the cell (Carmelo et al. 1997 ).Even though the activation of Pma1 at low pH is glucose-dependent due to the continuous r equir ement of cytosolic ATP, the mechanism of Pma1 activation in this condition apparently differs from the one observed for the well-described glucose-induced activation of Pma1 (Mazón et al. 2015 ).Pma1 activation by glucose metabolism is reversible and implicates a shift of the optimum pH to more alkaline v alues, wher eas activ ation in cells exposed to the weak or ganic acid, succinic acid, r esults in an irr e v ersible c hange , i.e .not as-sociated with an alteration of the optimum pH but is dependent on the growth phase (Eraso and Gancedo 1987, Benito et al. 1992, Mazón et al. 2015 ).Both these activ ation mec hanisms hav e been shown to result in an increase in affinity for ATP and to r equir e the C-terminal domain of Pma1 (Eraso and Gancedo 1987, Benito et al. 1992, Mazón et al. 2015 ).At low pH v alues, stim ulation of Pma1 is associated with a conformational change and potential destabilization of salt bridges between the R and P domains (Zhao et al. 2021 ).In S. cerevisiae , three phosphorylation residues within the R domain have been identified as r ele v ant, contributing to the r elie v e of autoinhibitory inter actions between the R and P domains: Ser899, whose phosphorylation correlates with a higher affinity of Pma1 for ATP; and Ser911 and Thr912, consisting of two adjacent residues whose phosphorylation leads to an increase in V max and is crucial for the full activation of Pma1 (Portillo 2000 , Eraso et al. 2006, Mazón et al. 2015 ).The phosphorylation state of these residues is so critical that mutations of PMA1 at these sites (S911A-T912A) can lead to a substantial reduction in Pma1 activity, impeding cell growth (Guarini et al. 2024 ).Specific yeast protein kinases play an instrumental role in the activation of Pma1 through phosphorylation.The protein kinase Hrk1 is a major acetic acid stress tolerance determinant in yeast (Antunes et al. 2018, 2023, Mira et al. 2010b, Bernardo et al. 2017, Guerr eir o et al. 2017, Xu et al. 2019a ), belonging to the NPR/Hal family (Antunes and Sá-Correia 2022 ).Under acetic acid stress conditions, Hrk1 contributes to the phosphorylation of Pma1 residues Ser911 and Thr912 (Guerr eir o et al. 2017 ), whereas in response to glucose metabolism the kinases Ptk1 and Ptk2 act both in a lar gel y r edundant manner in mediating the phosphorylation of Ser911 and Thr912, with Ptk2 promoting the double phosphorylation more ef-Figure 2. Illustration of the mechanisms of response and adaptation, reported and proposed for S. cerevisiae , to acetic acid stress through the regulation of ion homeostasis and fluxes.Some of these mechanisms are included in Table 1 .Kinases and phosphatases are displayed in yellow and pink, r espectiv el y.Tr anscription factors are displayed in purple.Membrane proteins reported to be associated with acetic acid stress tolerance are r epr esented in red.Pma1 is shown in the monomeric form to convey the mechanisms of regulation.Dashed arrows refer to putative or indirect associations.
ficiently than Ptk1 (Guarini et al. 2024 ).Although Hrk1 contributes to enhancing Pma1 activity under acidic conditions (Antunes et al. 2023 ), where it is presumed to be most active, its role in activating this proton pump in response to glucose metabolism appears to be minimal or undetectable (Goossens et al. 2000, Antunes et al. 2023, Guarini et al. 2024 ).Further investigation is needed to elucidate the putative roles of kinases Ptk1 and Ptk2, as well as to identify potential involvement of other kinases and the mechanisms underlying Hrk1 activity in the activation of Pma1 under acetic acid str ess.Inter estingl y, the activity of Pma1 stimulated by an increase in cytosolic H + concentration can trigger a signaling cascade leading to TORC1 activation (Guarini et al. 2024 ).For instance, the transport of amino acids via H + symporters, causing a local increase in cytosolic H + concentration, can stimulate Pma1 activity (Guarini et al. 2024 ).This enhanced activity might then contribute to TORC1 activation through mechanisms that are yet to be full y understood.Conv ersel y, the activ ation of TORC1 appears to feedback-inhibit Pma1 phosphorylation at the phosphorylation sites Ser911-Thr912 (Guarini et al. 2024 ).Notably, it has been proposed that Hrk1 might be involved in the stimulation of Ptk1 and Ptk2 in response to H + increase, based on the observation that the Pma1 residues Ser911 and Thr912 exhibit less pronounced phosphorylation upon H + -coupled amino acid uptake in the hrk1 deletion mutant strain, and that Hrk1 is unable to sustain the phosphorylation of these residues in ptk1 ptk2 cells (Guarini et al. 2024 ).Notably, the activity of Pma1 is reduced in vma m utants, r esulting either from Pma1 internalization, as a compensatory mechanism, or due to limitations in its activity through yet unidentified mechanisms (Kane 2016, Wilms et al. 2017 ).V -A TPase activity is crucial for yeast adaptation to acetic acid stress, as vma m utants ar e not able to r ecov er the initial pHi dr op induced by this acid and resume growth (Carmelo et al. 1997, Kawahata et al. 2006, Mira et al. 2010b, Tarsio et al. 2011, Wilms et al. 2017, Deprez et al. 2021, Mota et al. 2024 ).Ov er all, the coordination of Pma1 and V-ATPase activity represents a vital mechanism for maintaining pH homeostasis within the cell, in the absence or presence of acetic acid stress.

Initial pHi of individual cells influences yeast ability to adapt to sudden acetic acid stress
Cell-to-cell heterogeneity is a relevant parameter to consider in the context of a yeast cultur e ada ptation to sudden acetic acidinduced stress .T he ada ptation to acetic acid str ess is partiall y associated to the initial pHi of individual cells.Lo w er initial pHi values in a subpopulation of S. cerevisiae cells were reported to pr e v ent shar p dr ops in pHi following acetic acid exposure since the r esulting intr acellular concentr ation of dissociated acid is pr esumably lo w er in these cells, enhancing the likelihood of resum-Table 1. Brief descriptions of the mechanisms linked to the processes underlying acetic acid toxicity, as enumerated in Fig. 1 .These descriptions may also refer to mechanisms of response and adaptation to acetic acid, as depicted in Fig. 2 , where necessary, for clarity.

Process Mechanism References
( 1 ) Acetic acid passive diffusion Acetic acid in its undissociated form (when extracellular pH < pKa) crosses the PM through passive diffusion, likely adopting a dimeric structure Casal et al. ( 1996 ), Mollapour and Piper ( 2007 ) Exposure to acetic acid destabilizes the PM, disrupting pr oper selectiv e permeability.As a r esponse mec hanism, alterations of plasma membrane lipid composition restrict the passive diffusion of lipophilic toxic compounds into the cell (Fig. 2 ) Godinho et al. ( 2018 ), Ribeiro et al. ( 2022 ) ing pr olifer ation (Fernández-Niño et al. 2015 ).These mechanisms are still fairly uncharacterized, thus an explanation for this phenomenon may also be attributed to other intrinsic physiological factors, such as higher basal or acetic acid-induced H + -ATPase pumps activity, more efficient acetate efflux systems, or higher plasma membrane impermeability in those cells (Fernández-Niño et al. 2015 ).
The importance of the relationship between pH homeostasis and acetic acid tolerance is evidenced by the differences in shortterm alterations of pHi upon acetic acid stress exposure in other yeast species.Measurements of pHi in C. glabrata exposed to acetic acid stress revealed that this acid causes a similar decrease in pHi as the one reported in S. cerevisiae cells and that the recovery of pHi in both species is similar, following adaptation to acetic acid stress, although S. cerevisiae appears to be more tolerant to this stress condition (Ullah et al. 2013b ).Differences among C. glabrata str ains in toler ance to acetic acid str ess hav e been attributed to a higher activity of the plasma membr ane H + -ATP ase CgPma1 and a diminished accumulation of intracellular acetate (Cunha et al. 2017 ).The highly acetic acid tolerant yeast species, Zygosaccharomyces bailii , exhibits a remarkable tolerance to short-term decreases in pHi (Arneborg et al. 2000, Dang et al. 2012 ).This difference has been proposed to be a result of a high impermeability of Z. bailii plasma membrane to the undissociated acetic acid form, a high buffering capacity of Z. bailii cytosol, and/or a higher amount of energy reserves available for the stress response compared to S. cerevisiae (Arneborg et al. 2000 ).Furthermore, Z. bailii w as also sho wn to be able to tempor aril y toler ate a lar ge dr op in pHi induced by high inhibitory acetic acid concentrations during the exponential phase, which is only restored once cell proliferation ceases (stationary phase) (Dang et al. 2012 ).The high acetic acid tolerance phenotype of Z. bailii has been proposed to be in part due to the existence of a subpopulation of tolerant cells that have a basal lo w er pHi (from 0.4 to 0.8 pH units) than the acidsensitiv e bulk population, r esulting in a lo w er pr oportion of intr acellular dissociation of acetic acid (Stratford et al. 2013b ).

T he puta ti v e role of Snf1 kinase in acetic acid stress adaptation as an energy status-and pH-sensor
Cells lacking the SNF1 gene, encoding the Snf1 kinase, demonstrate significant sensitivity to acetic acid stress (Mira et al. 2010b ).T his kinase pla ys a crucial role in modulating the expression of genes responsible for the catabolism of alternative carbon sour ces in y east, particularly under conditions of glucose starvation (Simpson-Lavy andKupiec 2022 , 2023 ).Recent studies have expanded our understanding on Snf1's function in this yeast species, r e v ealing that, in addition to being an energy sensor, it also has a pH-sensing module, identified as a poly-histidine (poly-HIS) tract located in the prekinase region (Simpson-Lavy and Kupiec 2022 ).Full activation of Snf1 in response to glucose de pri vation is ac hie v ed thr ough thr ee independent mec hanisms: phosphorylation at the T210 r esidue, pr otonation of the polyHIS tract, and de-SUMOylation of the K549 residue (Simpson-Lavy and Kupiec 2022 ).Changes in pHi le v els dir ectl y influence the protonation state of the pol yHIS tr act; when pHi values become more alkaline, the pol yHIS tr act under goes depr otonation, wher eas acidic pHi values lead to its protonation, contributing to its activation (Simpson-Lavy and Kupiec 2022 ).The phosphorylation of Snf1 at T210 occurs, intriguingl y, e v en in the pr esence of glucose, when yeast cells are exposed to acetic acid stress (Mira et al. 2010b ), a phenomenon that could reflect Snf1's role in sensing the cell's energy status .T his hypothesis can be rationalized by considering that ADP is the k e y metabolite acti vating Snf1 under conditions of glucose depletion (Mayer et al. 2011 ).Under acetic acid str ess, ther e is an increase in the activity of plasma membrane H + -ATPase (Pma1) (Carmelo et al. 1997, Antunes et al. 2023 ), leading to heightened ATP consumption and potentially elevated ADP levels (Pampulha and Loureiro-Dias 2000 ).This suggests that acetic acid-induced ATP depletion and the presumable subsequent increase in ADP levels could serve as the primary driver behind Snf1 phosphorylation under these conditions.In addition to T210 phosphorylation, the drop in cytosolic pH caused by acetic acid stress is expected to lead to the protonation of the polyHIS tract, ther eby incr easing Snf1 activity.Ov er all, these observ ations suggest a complex interplay between Snf1 activity and acetic acid stress, pointing to a sophisticated regulatory network where Snf1 integrates signals from both the cellular energy state (phosphorylation of T210) and the pHi (pol yHIS pr otonation).Further r esearc h is necessary to fully elucidate these relationships and the precise mec hanisms thr ough whic h Snf1 coordinates cellular responses to acetic acid stress .T he role of Snf1 in the response and adaptation to acetic acid stress is pr obabl y not onl y limited to intr acellular H + concentration sensing, but also comprises the modulation of potassium and iron homeostasis, as outlined in the corresponding sections.

Homeostasis and regulation of potassium (K + ) fluxes and the response and tolerance to acetic acid stress
While Pma1 gr eatl y contributes to the regulation of pHi and plasma membrane potential, it is important to note that the pHi is determined by multiple factors beyond those described abo ve .
Various transporters located at the plasma membrane and organellar membranes contribute to the regulation of pHi (Yenush 2016, Ariño et al. 2019 ).Among these, alkali/cation exchangers play a significant role in facilitating the exchange of protons and other cations, helping to regulate and balance the pHi and plasma membrane potential (Cyert andPhilpott 2013 , Ariño et al. 2019 ).Potassium (K + ) is essential for various physiological processes in S. cerevisiae , including the maintenance of both pHi and plasma membr ane potential, whic h ar e crucial for enzyme function, and the maintenance of cell volume, osmotic stability, negativ e c har ge compensation, protein synthesis, and ov er all cellular functioning (Cyert andPhilpott 2013 , Yenush 2016 ).Potassium plays k e y roles in organelles; the redistribution of this ion intracellularly is crucial for proper homeostasis maintenance.Most of the intracellular potassium is accumulated in the v acuole, wher eas the concentration in the cytosol is rather low and k e pt constant (Herr er a et al. 2013 ).The intracellular potassium distribution is dependent on pHi, which under optimal conditions is close to neutral in the cytosol, while in organelles such as the vacuole , endosomes , and Golgi a ppar atus, it is r elativ el y mor e acidic compar ed to the cytosol, or slightly alkaline in the case of mitochondria (Orij et al. 2011, Ariño et al. 2019 ).Failure to maintain pH homeostasis has a substantial impact on the generation of the proton gradients r equir ed for proper potassium transport and distribution (Ariño et al. 2019 ).Disruptions in K + homeostasis can impact the distribution and balance of other ions and lead to cellular dysfunction, highlighting the importance of finely tuned regulatory mechanisms .For instance , potassium has been shown to affect copper and iron metabolism (Zhang et al. 2019 ), as described in the next section.The potassium homeostasis r egulation mec hanisms inv olve complex netw orks of transporters and sensors that adjust K + le v els in r esponse to envir onmental and internal cues, ensuring cellular adaptability under varying conditions, in particular under acetic acid stress.For instance, supplementation of K + in the cultivation medium was found to enhance tolerance to acetic acid stress in S. cerevisiae , Z. bailii , and Kluyveromyces marxianus (Macpherson et al. 2005, Mira et al. 2010b, Xu et al. 2019b, Castillo-Plata et al. 2022, Antunes et al. 2023 ).Additionall y, exposur e to weak acids such as acetic , propionic , butyric , and benzoic acids was found to increase potassium influx and intracellular accumulation (Ryan et al. 1971, Macpherson et al. 2005 ).
To maintain a balanced membrane potential, S. cerevisiae relies on the coordination of potassium uptake and efflux systems; disruption of high-affinity potassium uptake results in plasma membr ane hyper polarization, wher eas the absence of potassium efflux systems leads to plasma membrane depolarization (Kinclova-Zimmermannova et al. 2006, Nav arr ete et al. 2010 , Petr ezsél yov á et al. 2011 , Masaryk and Syc hr ov á 2022 ).Despite yeast cells accum ulating high concentr ations of potassium (200-300 mM), excessiv e intr acellular potassium le v els can hav e a negativ e physiological impact besides destabilization of plasma membrane potential, suc h as v acuole deacidification, and alter ations of cell volume and pHi (Cyert andPhilpott 2013 , Ariño et al. 2019 ).A compr ehensiv e ov ervie w of potassium uptake and efflux systems in se v er al yeast species is provided in (Ariño et al. 2019 ).In S. cerevisiae , the plasma membrane transporters Trk1 and Trk2 mediate high-affinity potassium uptake, which is the principal consumer of the electr oc hemical gr adient gener ated by Pma1 (Portillo et al. 2005, Barreto et al. 2011 ).Trk1 is the main player in potassium uptake, as evidenced by the prominent increase in potassium requirements and growth impairment observed upon its deletion.Inter estingl y, the potassium affinity of Trk1 changes gradually from high to lo w accor ding to intracellular and extracellular K + concentration (Masaryk and Sychrová 2022 ).In contrast, the contribution of Trk2 to K + acquisition is r elativ el y minor, potentiall y attributed to its lo w er expression levels (Ramos et al. 1994, Yenush 2016 ), but this transporter exhibits a relevant role in the maintenance of plasma membrane potential (P etrezsély ová et al. 2011 ).Trk2 was initially suggested to be involved in low affinity transport, but subsequent findings demonstrated its ability to mediate high/moderate affinity potassium uptake when expressed from a strong promoter (Ramos et al. 1994, Yenush 2016 ).Notably, Trk1 has been associated to S. cerevisiae 's tolerance to acetic acid stress, as demonstrated by the inability of cells lacking this transporter system to grow under these conditions (Kawahata et al. 2006, Mira et al. 2010b, Mota et al. 2024 ).Additionall y, m utations in TRK1 have been shown to impr ov e toler ance to acetic acid str ess (Xu et al. 2019b ).An inter esting featur e of Trk1 is its ability to mediate anion extrusion currents, in particular chloride (Cl − ), but also other anions such as I − , Br − , SCN − , or NO 3 − , whose order of selectivity changes with pH (Kuroda et al. 2004, Rivetta et al. 2011 ).The currents and permeability of anions such as formate and acetate, tested at pH 5.5, are much smaller than the aforementioned anions (Kuroda et al. 2004, Rivetta et al. 2011 ).The implications, if an y, this featur e might hav e in the r esponse and ada ptation to acetic acid stress are elusive.The regulation of Trk1 is complex (Ariño et al. 2019 , Masaryk and Syc hr ov á 2022 ) and has not been c har acterized under acetic acid stress.Under nonstressing conditions, it occurs mainly at the post-tr anslational le v el thr ough phosphorylation, with se v er al k e y players contributing to its activity and stability .Notably , the kinases Hal4 and Hal5, from the Npr/Hal family (Antunes and Sá-Corr eia 2022 ), ar e known for stabilizing Trk1 at the plasma membrane by phosphorylating its C-terminal end, a process that also involves the activity of the kinase Snf1 (Portillo et al. 2005, Pérez-Valle et al. 2007, Casado et al. 2010 ).The Snf1 kinase is notable for its influence on the activity of the Trk system (Portillo et al. 2005 ).Deletion of SNF1 results in several phenotypic effects, including reduced growth under potassium-limited conditions, decr eased potassium accum ulation, and hyper polarization of the plasma membrane (Portillo et al. 2005 ).These effects can be partially mitigated by overexpression of TRK1 and HAL5 (Portillo et al. 2005 ), wher eas ov er expr ession of SNF1 fails to suppress the growth defects of deletion mutants trk1 trk2 and hal4 hal5 .These observations suggest that Snf1, in its nonphosphorylated state, as it occurs in the presence of glucose, acts upstream of the kinases Hal4 and Hal5 and the potassium importer Trk1 (Portillo et al. 2005 ).T his implies that Snf1 pla ys a major role in modulating potassium uptake through a mechanism independent of the phosphorylation of T210.The phosphatase Ppz1 is implicated in the negativ e r egulation of Trk1 by dephosphorylation.This action of Ppz1 is r esponsiv e to c hanges in pHi; an incr ease in pHi leads to Trk1 dephosphorylation, whereas a decrease in pHi triggers the interaction between Ppz1 and Hal3 (its main regulator), forming a complex that effectiv el y inhibits Ppz1's activity, resulting in the alleviation of the repression of Trk1 (Yenush et al. 2005, Albacar et al. 2022 ), consistent with an increase of its activity under weak acid stress (Xu et al. 2019b ).ARL1 , encoding a conserved guanine nucleotide-binding protein, has been identified as a determinant of acetic acid str ess toler ance based on susceptibility phenotype observed in the deletion mutant strain (Kawahata et al. 2006, Mira et al. 2010b, Mota et al. 2024 ).Arl1 was initially proposed to act upstream of the kinases Hal4 and Hal5 in potassium uptake (Munson et al. 2004 ).Ho w e v er, subsequent studies hav e shown that deletion of ARL1 does not affect Trk1's intracellular localization (Munson et al. 2004, Pérez-Valle et al. 2007 ), suggesting an alter-nativ e mec hanism of action.Arl1 is known to play a r ole in intr acellular tr affic king (Munson et al. 2004 ), which may be r ele v ant in the context of acetic acid adaptation alongside the modulation of potassium fluxes.Ho w e v er, the pr ecise r oles of Arl1 in potassium uptake and acetic acid tolerance require further elucidation.
Potassium export is primarily ensured by three transport systems , Nha1, Ena ATPases , and Tok1 (Ariño et al. 2019 ).Tok1 is an outw ar d-r ectifier c hannel specific for potassium, whic h plays an important role in the maintenance of plasma membrane potential following depolarization (Mar esov a et al. 2006 ).Ena enzymes ar e P-type ATP ases that couple the hydr ol ysis of ATP to the export of alkali metal cations .T hey ar e usuall y found in low le v els under basal cultivation conditions and are weakly expressed at low pH (Ruiz and Arino 2007 ), thus they may not contribute substantially in the adaptation to weak acid-induced stress.Nha1 is a H + /K + ,Na + antiporter, being the plasma membrane potassium efflux system most biologicall y r ele v ant for gr o wth under lo w pH conditions.It is a housek ee ping protein produced in low amounts and regulated at the post-translational level, whose main function is the detoxification of surplus alkali metal cations (Kinclová et al. 2001 ).It contributes to the r egulation of plasma membr ane and has a minor but significant role in the maintenance of pHi homeostasis (Syc hr ov á et al. 1999 , Kinclov a-Zimmermannov a et al. 2006 ).Nha1 and the endosomal K + ,Na + /H + antiporter Nhx1 are independentl y r esponsible and contribute equally for the efflux of potassium from the cytosol preventing overaccumulation of surplus potassium cations (Brett et al. 2005 ).Both the Nha1 and Nhx1 exc hangers ar e determinants of tolerance to acetic acid stress and ar e r equir ed for cell gr o wth at lo w pH values (Bañuelos et al. 1998, Brett et al. 2005, Mira et al. 2010b, Antunes et al. 2023 ).Mor eov er, Nha1 is a putative phosphorylation target of the Hrk1 kinase.Under acetic acid stress conditions, Nha1 has been proposed to have a r ele v ant r ole in the maintenance of plasma membr ane potential (Antunes et al. 2023 ).Ho w e v er, the mec hanisms underl ying its molecular function in acetic acid stress tolerance are still uncovered.Nhx1 has a dominant role relative to Nha1 in the modulation of pHi, by opposing the activity of the V-type H + -ATPase to alkalinize cellular compartments (Brett et al. 2005 ).The role of Nhx1 is not limited to ion homeostasis but also includes v esicle tr affic king from the endosome, as alterations in pHi substantially impact or ganellar mor phology and v esicular tr affic king (Br ett et al. 2005 ).

Acetic acid stress alters intracellular iron (F e 2 + /F e 3 + ) availability
Metal metabolism undergoes significant alterations in yeast cells exposed to acetic acid stress .T his stress condition disrupts the homeostasis of metal ions, particularly affecting iron levels and the expression of genes involved in iron uptake and metabolism, including the Aft1 tr anscription factor, whic h plays a major role in regulating the iron regulon under iron limitation conditions (Kawahata et al. 2006 ).Inter estingl y, a study has shown that under acetic acid stress, Aft1 exhibits increased transcription levels and is translocated to the nucleus, suggesting its activation (Kawahata et al. 2006 ).Ho w e v er, contr astingl y, intr acellular ir on concentr ations incr ease nearl y 2-fold upon exposur e to acetic acid stress (30 min), and abolition of the expression of genes involved in iron uptake exacerbates acetic acid sensitivity, e v en though supplementation with iron (1-100 μM of FeSO 4 ) a ppar entl y does not alleviate acetic acid stress sensitivity.These seemingly contradic-tory findings may be consistent by considering that Aft1 activation does not depend on cytosolic ir on le v els but on the iron-sulfur (Fe-S) cluster (ISC) mac hinery and that ISCs ar e particularl y vulner able to o xidati v e str ess, as their oxidation leads to loss of protein function and release of free iron (Lill et al. 2012 ).A decrease in ISC protein synthesis within mitochondria can activate the iron regulon e v en under ir on-r eplete conditions (Lill et al. 2012 ), indicating that acetic acid stress might cause a reduction in ISCs' availability.T hus , a possible regulation of iron uptake and metabolism, in response to a high sensitivity of ISCs to acetic acid-induced oxidativ e str ess, is suggested since ir on-containing pr oteins ar e involved in the o xidati ve stress response and iron excess may gener ate dama ging oxygen r adicals (Matsuo et al. 2017 ).
The protein kinase Snf1 activity is linked to ISCs sufficiency; a mechanism that ensures the inhibition of respiratory enzymatic activity in the absence of glucose when iron scarcity leads to insufficient availability of ISCs (Simpson-Lavy and Kupiec 2023 ).When there is competition among se v er al cellular pr ocesses that r equir e ir on for pr oper function, the n uclear acti vity of Snf1 is inhibited, while its cytosolic activities remain unaffected.This inhibition is exerted by interaction of the protonated polyHIS tract of Snf1 with Aft1, which results in a decrease of nuclear Snf1 activity by half (Simpson-Lavy and Kupiec 2023 ).Under acetic acid stress conditions, it can be speculated that Snf1 exhibits a protonated pol yHIS tr act due to intr acellular acidification, pr omoting its interaction with the nucleus-localized Aft1, thus resulting in inhibition of nuclear Snf1 activity (Simpson-Lavy and Kupiec 2023 ).The protein kinase Hog1, recognized as playing a role in acetic acid stress tolerance in S. cerevisiae and Candida glycerinogenes (Mollapour and Piper 2006, Mira et al. 2010b, Ji et al. 2016, Guar a gnella et al. 2019, Ye et al. 2022, 2023 ), is r a pidl y activ ated through phosphorylation in response to this stress in S. cerevisiae (Mollapour and Piper 2006 ).Hog1 directly phosphorylates Aft1 at residues S210 and S224 (Martins et al. 2018 ), presumably leading to its reduced activity by promoting its nuclear export (Ueta et al. 2007 ).Ho w e v er, Aft1's activity is not solely dependent on phosphorylation; its interaction with the monothiol glutaredoxins Grx3/4 is crucial for dissociating Aft1 fr om tar get pr omoters when ir on le v els ar e sufficient.This cr eates a scenario wher e cells with activated Hog1 might still retain active Aft1 (Martins et al. 2018 ).
Ther efor e, it is plausible to speculate that acetic acid exposure could impair the activity of Grx3/4, which require Fe-S clusters as cofactor, and/or disrupt the interaction between Hog1 and Aft1, resulting in nuclear-localized active Aft1, and consequently, alterations in iron levels .Nevertheless , the mechanisms underlying the activation of Aft1, its interaction with Snf1 and Hog1, and the role of iron in yeast response and adaptation to acetic acid stress, require further studies.
Iron metabolism is intricately linked to potassium compartmentalization within organelles .P otassium uptake via the trans-Golgi network K + /H + exchanger Kha1 is crucial for improving iron acquisition (Zhang et al. 2019 ).This pr ocess occurs thr ough copper insertion into the m ulticopper ferr oxidase a poFet3.Evidence supporting this includes observations that deletion of KHA1 results in r espir atory deficiency, a phenotype r escued by ir on supplementation (Zhang et al. 2019 ).Mor eov er, the deletion of the potassium exchangers Vnx1 (vacuolar) or Nhx1 (late endosomal) results in cytosolic potassium accumulation, thereby facilitating copper loading to apoFet3 by increasing the K + supply for Kha1.Conv ersel y, deletion of the mitochondrial potassium transporter Mdm38 diminishes cytosolic potassium accum ulation, r esulting in reduced Golgi potassium levels and subsequent iron deficiency (Zhang et al. 2019 ).
In C. albicans , exposure to different weak acids, including acetic acid at pH 5.5, leads to ele v ated tr anscript le v els of genes associated with iron homeostasis.Ho w ever, contrasting with S. cerevisiae , a substantial reduction in intracellular iron levels is observed under acetic acid stress (4 h of exposure) (Cottier et al. 2015 ).This occurs despite the absence of iron limitation in the growth medium.Ho w e v er, enhancing ir on uptake in C. albicans does not alleviate the reduction in intracellular iron levels or mitigate growth inhibition under weak acid stress conditions (Cottier et al. 2015 ).This suggests that the reduced intracellular iron level may not mainly occur due to impairment of iron uptake systems.

Zinc supplementation alleviates yeast growth inhibition under acetic acid stress
Zinc (Zn 2 + ) supplementation of the cultivation medium has emerged as a promising strategy to mitigate the inhibitory effects of acetic acid stress (Wan et al. 2015, Zhang et al. 2017, Chen et al. 2022 ).Zinc is an essential nutrient r equir ed for the structure and function of various proteins (Eide 2006 ).Addition of zinc sulfate to the culture medium of yeast cells exposed to acetic acid stress leads to increased levels of amino acids such as alanine, valine , and serine , with alanine accumulation being associated with enhanced glucose consumption and reduced accumulation of reacti ve o xygen species (ROS) and to the incr ease of intr acellular glutathione (GSH), pr esumabl y as a r esponse to o xidati v e str ess (Wan et al. 2015 ).Zinc plays an essential role as a cofactor for pr oteins, including alcohol dehydr ogenases (ADHs) the Cu, Zn super oxide dism utase (SOD) Sod1 and se v er al tr anscription factors (Eide 2006 ).For instance, zinc is crucial for the proper functioning of the Haa1 transcription factor (Kim et al. 2019 ), as detailed in the next section.Under acetic acid stress, zinc supplementation leads to increased transcription levels of genes involved in ergosterol biosynthesis, accompanied by elevated ergosterol levels, and to decreased transcription levels of genes encoding acetate uptake tr ansporters, suc h as Ady2, Ato2, and Jen1 (Zhang et al. 2017 ).

Toxicity and adapti v e responses to intr acellularl y accumula ted aceta te
Acetate is a by-product of yeast fermentation but can also serve as a carbon source.Under conditions of acetic acid stress, acetate accumulates within the cell, as explained abo ve .T he intracellular accumulation of acetate can disrupt cellular metabolism, leading to increased turgor pressure and significant o xidati ve stress (Pampulha and Loureiro-Dias 1990 , Piper et al. 2001, Giannattasio et al. 2005 ).The presence of high acetate concentrations elevates the activity of antioxidant enzymes such as SOD and catalase, along with increasing levels of o xidati vely modified proteins (Semch ysh yn et al. 2011 ).Interestingly, as the result of preincubation with low concentrations of hydrogen peroxide, yeast cells can acquir e cr oss-pr otection a gainst acetic acid str ess thr ough the action of the transcription factor Yap1, underscoring the prooxidant effects of acetate (Semch ysh yn et al. 2011 ).A Candida krusei strain with improved acetic acid tolerance obtained through genome shuffling w as sho wn to display incr eased le v els of intracellular catalase activity, also indicating that the accumulation of acetate results in o xidati ve stress in this species (Wei et al. 2008 ).The accumulation of acetate within the cell leads to an increase in misfolded pr oteins, adv ersel y affecting their processing in the endoplasmic reticulum (ER).This can disrupt the synthesis of secr etory and tr ansmembr ane pr oteins, including m ultidrug r esistance (MDR) transporters (Kawazoe et al. 2017 ).In response to the ele v ated intr acellular acetate le v els and the r esultant str ess on protein folding in the ER, yeast cells activate the unfolded protein response (UPR) (Kawazoe et al. 2017 ).The UPR is a critical cellular mechanism to restore ER normal function by reducing the accumulation of misfolded proteins.It involves the Ire1 transmembr ane RNase, whic h senses misfolded pr oteins in the ER (Kawazoe et al. 2017 ).Upon activ ation, Ir e1 triggers the splicing of the tr anscription factor HAC1 mRNA.Hac1 then enters the nucleus, where it upregulates the expression of genes involved in protein folding, ER-associated degradation (ERAD), and ER stress response (Kawazoe et al. 2017 ).

Carboxylic acid uptake transporters mediate deleterious acetate uptake
The uptake of acetate in S. cerevisiae is facilitated through the carboxylic acid transporters Ady2 (Ato1), Ato2 (Fun34), and Jen1 (Casal et al. 2016, Zhang et al. 2017 ).Deletion of genes encoding these transporters, in particular ADY2 and ATO2 , results in impr ov ed gr owth under acetic acid str ess (Gentsc h et al. 2007, Zhang et al. 2017 ).Additionall y, intr acellular acetate accum ulation in the ady2 deletion mutant was found to be reduced, suggesting that e v en in conditions where the extracellular pH is below acetic acid pKa, there may be some acetate uptake.The cytosolic accumulation of acetate is a signaling mechanism for the activation of the transcription factor Haa1 (Kim et al. 2019 ).This transcriptional regulator is the major activator of the transcription of acetic acidr esponsiv e genes in S. cerevisiae , C. glabrata , and Z. bailii (Mira et al. 2010b, Bernardo et al. 2017, Antunes et al. 2018 ).The function of Haa1 in S. cerevisiae was shown to be dependent on the acetate binding to the N-terminal r egion, whic h conv erts Haa1 to an active form, likel y thr ough a conformational c hange, r endering it ca pable of DNA binding to target gene promoters (Kim et al. 2019 ).

Acetate acti v e export through MDR transporters
The ada ptiv e r esponse to acetic acid str ess in S. cerevisiae involves the active export of acetate through specific plasma membr ane tr ansporters (Tenr eir o et al. 2000, 2002, Dos Santos et al. 2014, Sá-Correia and Godinho 2022, Zhang et al. 2022 ).These tr ansporters, implicated in m ultidrug/m ultixenobiotic r esistance (MDR/MXR) belong to the major facilitator superfamily (MFS) or to the ATP-binding cassette (ABC) superfamily (Sá-Correia and Godinho 2022 ).The MFS transporters are secondary active transporters of small solutes , including sugars , amino acids , and drugs, acr oss biological membr anes using the ener gy stor ed in the electr oc hemical gr adients (P ao et al. 1998 ).On the other hand, the ABC superfamil y tr ansporters utilize the ener gy of ATP hydr ol ysis to transport both small molecules and macromolecules acr oss biological membr anes , pla ying critical r oles in cellular pr ocesses such as nutrient uptake, drug resistance, and detoxification (Pao et al. 1998 ).MDR/MXR transporters are documented as drug/xenobiotic efflux pumps for their hypothesized ability to catalyze the efflux of multiple cytotoxic compounds, thus contributing to the acquisition of stress tolerance in yeast cells.Howe v er, recent studies suggest that some MDR transporters may exert their effects indir ectl y, by modulating membr ane potential and/or pHi, rather than through the direct export of cytotoxic compounds (Dos Santos et al. 2014 ).The physiological substrates for some of these transporters have been proposed, including the transport of the acetate metabolite (Fernandes et al. 2005, Zhang et al. ( 2022 ).
Among the various MFS transporters, some have been identified as determinants of tolerance to inhibitory compounds found in industrial pr ocesses, whic h tr anslates to their ability to utilize the proton motive force to extrude toxic compounds from the cell in exchange for protons (Sá-Correia and Godinho 2022 ).Of these, Aqr1, Azr1, Tpo2, and Tpo3 have been shown to be determinants of acetic acid stress tolerance in S. cerevisiae (Tenreiro et al. 2000, 2002, Sá-Correia and Godinho 2022, Zhang et al. 2022 ).Deletion of the TPO2 and TPO3 genes in S. cerevisiae results in a significant incr ease in intr acellular acetate accum ulation, supporting the putativ e involv ement of Tpo2 and Tpo3 in acetate efflux (Fernandes et al. 2005, Zhang et al. 2022 ).Both TPO2 and TPO3 genes are targets of the transcription factor Haa1 under acetic acid stress .T he regulation of Tpo3 activity may involve Hrk1 since cells lacking this kinase exhibit increased acetate accumulation, and Tpo3 is a potential phosphorylation target of Hrk1 (Mira et al. 2010a, Guerr eir o et al. 2017 ).Ho w e v er, this r elationship r emains to be elucidated.In C. glabrata , the transporters CgTpo3 and CgAqr1 are reported determinants of tolerance to acetic acid stress (Costa et al. 2013, Bernardo et al. 2017 ).Similarly to the S. cerevisiae homologues, CgTpo3, but not CgAqr1, is involv ed in the r eduction of intr acellular acetate (Tenr eir o et al. 2002, Fernandes et al. 2005, Costa et al. 2013, Bernardo et al. 2017 ).CgAqr1 appears to be functionally like ScAqr1 and able to complement the acetic acid stress susceptibility phenotype of the S. cerevisiae aqr1 deletion mutant strain, suggesting an indirect role of CgAqr1 in acetic acid stress adaptation (Costa et al. 2013 ).Additionally, the transporter CgDtr1 from C. glabrata was identified as playing a role in pathogenesis and, as opposed to S. cerevisiae ScDtr1, contributes to acetic acid tolerance, functioning as a plasma membrane acetate exporter (Romão et al. 2017 ).
Regarding the ABC transporters involved in acetic acid tolerance, Pdr18 is the most well-c har acterized (Ribeir o et al. 2022 ).It plays a crucial role in actively incorporating ergosterol and maintaining maximum plasma membrane ergosterol content, especiall y to counter act the acetic acid str ess-induced decr ease of ergosterol content and lipid order (Godinho et al. 2018, Ribeiro et al. 2022 ).Pdr18 activity contributes to stabilizing plasma membrane potential and mitigate the increase in acetic acid-induced nonspecific membrane permeability (Godinho et al. 2018 ).Pdr12 is gener all y unr esponsiv e to se v er al str ess factors, but it shows a strong affinity to w ar d moder atel y lipophilic weak acids, pr oviding tolerance to sorbic and benzoic acid, but not for short-chain fatty acids, acetic or formic acids (Nygård et al. 2014 ).Inter estingl y, deletion of PDR12 in S. cerevisiae was shown to impr ov e acetic acid str ess toler ance, wher eas its ov er expr ession r esults in heightened sensitivity (Nygård et al. 2014 ).This phenomenon was considered the result of a higher conservation of ATP for other cellular processes when PDR12 expression is abrogated (Nygård et al. 2014 ).

Influence of plasma membrane lipid and pr otein alter a tions on ion flux es under acetic acid stress
One of the k e y adaptation mechanisms to acetic acid stress in y easts inv olv es the modification of plasma membr ane lipid composition.The rate at which the undissociated form of acetic acid diffuses into yeast cells is notably fast and is a process independent of the total concentration of acid (Lindahl et al. 2018 ).Ho w e v er, the pr esence of compounds in the culture medium that can partition into the plasma membr ane, suc h as liposoluble compounds (e.g.octanoic and decanoic acids) and alcohols (e.g.ethanol and n -butanol) substantially impact the plasma membrane spatial organization and the diffusion rate of acetic acid and other weak acids (Viegas et al. 1989, Alexandre et al. 1996, Casal et al. 1998, Lindahl et al. 2018 ).In the case of ethanol, an inhibitory product of alcoholic fermentation, significant changes occur in plasma membrane physical properties, including an increase in the area per lipid molecule and a decrease in both membr ane thic kness and order, thus facilitating a faster diffusion of acetic acid across plasma membrane (Lindahl et al. 2018 ).Consequently, cells exposed to acetic acid in the presence of ethanol exhibit decreased specific growth rates and an extended lag phase of gr owth r esultant fr om incr eased acetic acid diffusion rate, underscoring the combined toxic effects of ethanol and acetic acid on yeast cells (Lindahl et al. 2018 ).
The alteration of plasma membrane lipid composition constitutes an ada ptiv e r esponse to maintain plasma membr ane physicoc hemical pr operties suc h as fluidity, thic kness, permeability, and electr oc hemical potential under stress conditions (Lindberg et al. 2013, Lindahl et al. 2016, Godinho et al. ( 2018 ).The reduction in er goster ol le v els (Lindber g et al. 2013, Godinho et al. 2018 ) and activation of sphingolipid synthesis (Lindberg et al. 2013, Guerr eir o et al. 2016 ) has been observed in S. cerevisiae under acetic acid stress .T he pr otectiv e r ole of er goster ol a gainst acetic acid str ess pr esumabl y depends on its ability to limit the diffusion of acetic acid across the plasma membrane, a property that has been demonstrated in experiments using liposomes (Ferraz et al. 2023 ).Ho w e v er, it should be noted that the efficacy of er goster ol in reducing the permeability of plasma membrane to acetic acid is not only dependent on its concentration but also on the ov er all lipid composition of the plasma membrane (Gabba et al. 2020, Ferraz et al. 2023 ).The reported alterations in ergosterol concentration from yeast cells exposed to acetic acid varies, with studies reporting both decreases (Lindberg et al. 2013, Godinho et al. 2018 ) and increases (Guo et al. 2018 ) compared to unstressed cells .T his variability highlights the complexity of sterol metabolism in response to environmental stresses, suggesting that culturing conditions, y east gro wth phase, and le v el of acetic acid str ess hav e a significant impact.Despite these alterations in er goster ol content under acetic acid stress, genes involved in ergosterol biosynthesis exhibit increased transcription levels and have been identified as determinants of tolerance to this stress (Mira et al. 2010a,b , Godinho et al. 2018, Guo et al. 2018, Zhang et al. 2017, Mota et al. 2024 ).Furthermor e, incr ease in the tr anscription le v els of the ABC tr ansporter PDR18 wer e shown to be coordinated with an increase of transcripts of genes involved in ergosterol biosynthesis, underscoring its r ele v ance in the maintenance of physiological le v els of er goster ol under acetic acid stress (Godinho et al. 2018 ).
The regulation of the sphingolipids' biosynthetic pathway and sphingolipid content in S. cerevisiae and Z. bailii holds great influence on the tolerance to acetic acid stress (Guerreiro et al. 2016, Lindahl et al. 2016 ).In S. cerevisiae , proper regulation of sphingolipid metabolism is crucial for the activities of the plasma membr ane and or ganellar H + -ATP ases, as well as ir on homeostasis (Tani and Toume 2015 , Martins et al. 2018, Zhao et al. 2021 ).
As pr e viousl y mentioned, Pma1 localizes in sphingolipid-rich microdomains in the plasma membrane (Zhao et al. 2021 ).Additionally, deletion of genes involved in sphingolipid biosynthesis ( ELO3 , ORM1 , and ORM2 ) leads to the reduction of V -A TPase activity (Tani and Toume 2015 ).Furthermore, deletion of ISC1 , encoding a phospholipase involved in sphingolipid catabolism, results in the upregulation of the iron regulon, improper activation of Aft1, intracellular iron accumulation, and improved stress tolerance to high acetic acid concentrations (Rego et al. 2012, Martins et al. 2018 ).
Alterations in the levels of sphingolipid and its precursors impacts cellular processes such as cell fate, the activity of kinases and phosphatases, and pr otein tr affic king (Rego et al. 2018 ).The activation of Ypk1, encoding a TORC2 activated protein kinase r equir ed for plasma membrane lipid and protein homeostasis, under acetic acid str ess, r esults in the phosphorylation of several k e y pr oteins involv ed in sphingolipid biosynthesis (Guerr eir o et al. 2016 ).P articularl y, Ypk1 phosphorylates Orm1 and Orm2, whic h normall y inhibit the enzyme complex l -serine:palmitoyl-CoA acyltr ansfer ase, the first committed step in sphingolipid biosynthesis .T his phosphorylation r elie v es the inhibition on lserine:palmito yl-CoA ac yltr ansfer ase, thus incr easing flux into the sphingolipid pathway (Guerr eir o et al. 2016 ).Additionally, Ypk1 phosphorylates Lac1 and Lag1, two functionally redundant isoforms of the ceramide synthase complex, diverting long-chain base (LCB) precursors more efficiently into ceramides for complex sphingolipid production under acetic acid stress (Guerreiro et al. 2016 ).Additionall y, exposur e to high acetic acid concentrations induces the translocation of Isc1 from the ER to the mitoc hondria, whic h pr omotes an incr ease in cer amide le v els thr ough the hydr ol ysis of complex lipids inducing mitochondria-mediated regulated cell death (Rego et al. 2018 ).T herefore , the tight balance of sphingolipid metabolism is crucial for the adaptation to acetic acid stress.In Z. bailii , a m uc h higher basal concentration of complex sphingolipids than S. cerevisiae is found at the plasma membr ane, whic h incr eases upon acetic acid str ess exposur e and translates to a higher impermeability to acetic acid (Lindberg et al. 2013, Lindahl et al. 2016 ).Underscoring the r ele v ance of this ada ptation mec hanism for yeast toler ance to acetic acid stress, a C. krusei strain with improved tolerance to acetic acid was reported to exhibit higher plasma membrane impermeability (Wei et al. 2008 ).

Conclusions and outlook
The response and adaptation to sublethal concentrations of acetic acid involves the complex coordination of mechanisms aimed at maintaining the physiological balance of asymmetric concentrations of various ions .T his regulation is mediated by the coordinated activity of se v er al membr ane pr oteins, including the H + pumps Pma1 and V -A TP ase, alkali-cation exc hangers, and acetate exporters, in addition to the remodeling of plasma membrane lipid and protein composition (Fig. 2 ).
Manipulation of ion concentr ations, suc h as r eported for K + or Zn 2 + supplementation in the growth medium (Macpherson et al. 2005, Mira et al. 2010b, Wan et al. 2015, Zhang et al. 2017, Xu et al. 2019b, Castillo-Plata et al. 2022, Chen et al. 2022, Antunes et al. 2023 ), emerges as a potential strategy that can be explored for enhancing yeast tolerance to acetic acid stress.Genetic engineering aimed at altering the composition of plasma membrane lipids and/or proteins has been explored as an approach to improve tolerance to acetic acid stress.For instance, ov er expr ession of PMA1 and mutations in TRK1 have been demonstrated to confer impr ov ed toler ance to this stress (Lee et al. 2017, Xu et al. 2019b ) and ov er expr ession of genes involved in the synthesis and elongation of unsaturated fatty acids can increase the unsaturation index of fatty acids in the plasma membrane and elevate oleic acid levels in the cell, thereby providing protection against acetic acid stress (Zheng et al. 2013, Guo et al. 2018 ).Another study has attempted to incr ease the le v els of complex sphingolipids in the plasma membr ane thr ough modulation of gene expr ession r elated to the pr oduction of LCB and v ery-long-c hain fatty acids, as well as the conv ersion of cer amides into complex sphingolipids, but the ap-pr oac hes used have not yielded the desired outcomes (Lindahl et al. 2017 ).This underscor es the complexity of the mec hanisms underlying the regulation of sphingolipid metabolism and highlights the current challenges associated with engineering membrane lipid composition.
Understanding the mechanisms underlying tolerance to acetic acid str ess, particularl y concerning ion fluxes and balances, is crucial not only for developing more robust industrial yeast strains but also for identifying potential antifungal targets to mitigate the pr olifer ation of drug-r esistant pathogenic yeast str ains.Future studies aimed at exploring the intricate mechanisms governing ion homeostasis and acetic acid tolerance in clinically relevant yeast species will be fundamental to de v elop effectiv e antifungal ther a pies, giv en that numer ous antifungal a gents tar get ion homeostasis disruption and acetic acid has been implicated in the synergistic sensitization effects of azoles on Candida species (Moosa et al. 2004, Cunha et al. 2017, Li et al. 2018, Lourenco et al. 2019 ).Furthermore, delving into these mechanisms underlying ion homeostasis regulation and acetic acid stress tolerance in pr omising nonconv entional yeast species of biotec hnological r ele v ance, suc h as Yarrowia lipolytica and Rhodotorula toruloides (Mota et al. 2022 ) could guide the de v elopment of superior yeast cell factories enhancing the sustainability , productivity , and efficiency of industrial biopr ocesses, pr omoting a circular bio-based economy.

Figure 1 .
Figure 1.Illustration of the mechanisms of toxicity induced by acetic acid in S. cerevisiae .Brief descriptions of each of the enumerated acetic acid toxicity processes are provided in Table1.